Novel plate reader-based assay measuring glioprotection in primary adult optic nerve head astrocytes.

Abstract

Purpose: Optic nerve head (ONH) astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head, where they are the largest contributor to extracellular matrix synthesis during development and throughout life. In glaucoma, optic nerve head astrocytes (ONHAs) undergo significant pathological changes, resulting in activation and extracellular matrix remodeling. ONHAs contribute to the initiation of axon damage, given their sensitivity and response to mechanical and oxidative stress during disease pathogenesis. Furthermore, ONHAs are important for the maintenance of retinal ganglion cell physiology and function. Glioprotective strategies targeting the maintenance of normal ONHA structure and function in order to slow glaucoma pathology are thus of high clinical relevance.

Methods: We developed 96-well plate-based cell viability assays for primary culture of adult rat ONHAs. Assays determining cellular viability (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] live cell staining), late stage apoptosis (lactate dehydrogenase [LDH] release), and intracellular redox state (6-carboxy-2’, 7’ dichlorodihydrofluorescein diacetate [DCFDA] live cell staining) assays were employed to quantify the effect of the prototypic antioxidant, Trolox, in response to exogenously applied oxidative stress using tert-butylhydroperoxide (tBHP) as a model of disease-mediated oxidative stress.

Results: Levels of oxidative stress were increased in response to exogenously applied tBHP, as assessed by DCFDA fluorescence. Normalized DCFDA fluorescence showed a maximal 5.1-fold increase. The LD₅₀ for tBHP was 241 ± 20 µM in the LDH assay and 194 ± 5 µM in the MTT assay. 100 μM Trolox decreased the sensitivity of ONHAs to tBHP, shifting the LD₅₀ significantly. LD₅₀ values were 396 ± 12 µM in the LDH release and 383 ± 3 µM in the MTT assay. Vehicle treatment with 0.1% v/v ethanol did not significantly affect cellular responses to tBHP.

Conclusions: Antioxidant treatment increases ONHA viability and reduces the deleterious effects of oxidative stress. Our data provide important feasibility data for utilizing primary rat ONHAs in plate reader-based assays assessing novel therapeutics for glioprotection in glaucoma and other disorders affecting the optic nerve and ONH. Conversion of these standardized protocols to high-throughput testing strategies is highly feasible.